In these studies, we will determine the role of vitamin B12 in regulating nuclear de novo dTMP biosynthesis, maintaining genome stability, as well as elucidate the mechanisms underlying vitamin B12 in birth defects. Impairments nuclear in de novo dTMP biosynthesis underlie folate-associated pathologies in a gene by diet manner, as Shmt1-/+ mice fed a folate-deficient diet accumulate uracil in DNA and are sensitized to developing neural tube defects (NTDs) and colon cancer. Recently, SHMT was identified as a scaffolding protein required for proper assembly of the de novo dTMP biosynthesis complex at sites of DNA replication and repair. Vitamin B12 deficiency results in elevated intracellular 5-methylTHF and uracil accumulation in DNA, and has been suggested but not proved to cause NTDs. These proposed studies will determine the impact of a vitamin B12 deficiency on de novo dTMP biosynthesis and establish whether impairments in this pathway contribute to vitamin B12-associated NTDs. Furthermore, the findings from these studies will inform future clinical practice through identification of additional periconceptional measures to further prevent occurrence of prenatal folate - and vitamin B12-associated pathologies, and inform policy-making decisions in the development of population- based approaches to improve vitamin B12 status through mandated fortification. The two related and overlapping areas to be investigated are: Aim I. a) Determine if vitamin B12 deficiency impairs de novo thymidylate (dTMP) biosynthesis in the nucleus. These studies will determine the potential mechanisms by which vitamin B12 deficiency, as modeled by MTR inhibition leading to an irreversible accumulation of 5-methylTHF, impairs nuclear de novo dTMP biosynthesis via the: 1) disruption of the multi-enzyme complex for dTMP synthesis or 2) depletion of tetrahydrofolate cofactors required for nucleotide biosynthesis. b) Determine if pyrimidine nucleosides, dU and U, are differentially incorporated as either dUTP or dTTP in nuclear DNA. These studies will establish the origin(s) of uracil in nuclear DNA in a cell model of vitamin B12 deficiency, and reconcile the current uncertainty in the literature with respect to the nucleosides that are incorporated as dUTP. Aim II. Determine if vitamin B12 deficiency increases the incidence of NTDs in mouse models of impaired de novo dTMP biosynthesis. These studies will determine if maternal vitamin B12 deficiency increases NTD incidence in offspring through a gene by diet interaction using our mouse model of impaired de novo dTMP biosynthesis. Additionally, these studies will determine if vitamin B12 deficiency compromises genome integrity in maternal and fetal genomes.